Discovery of Novel Human Constitutive Androstane Receptor Agonists with the Imidazo[1,2-a]pyridine Structure

The nuclear constitutive androstane receptor (CAR, NR1I3) plays significant roles in many hepatic functions, such as fatty acid oxidation, biotransformation, liver regeneration, as well as clearance of steroid hormones, cholesterol, and bilirubin. CAR has been proposed as a hypothetical target receptor for metabolic or liver disease therapy. Currently known prototype high-affinity human CAR agonists such as CITCO (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime) have limited selectivity, activating the pregnane X receptor (PXR) receptor, a related receptor of the NR1I subfamily. We have discovered several derivatives of 3-(1H-1,2,3-triazol-4-yl)imidazo[1,2-a]pyridine that directly activate human CAR in nanomolar concentrations. While compound 39 regulates CAR target genes in humanized CAR mice as well as human hepatocytes, it does not activate other nuclear receptors and is nontoxic in cellular and genotoxic assays as well as in rodent toxicity studies. Our findings concerning potent human CAR agonists with in vivo activity reinforce the role of CAR as a possible therapeutic target.


■ INTRODUCTION
The constitutive androstane receptor (CAR, NR1I3) is a ligand-activated transcription factor belonging to the nuclear receptor subfamily NR1I.
Human CAR is dominantly expressed in hepatocytes. While the endogenous ligands of human CAR are obscure, a number of naturally occurring steroids such as androstanol, androstenol, and 5β-pregnane-3,20-dione have been proposed as endogenous inverse agonists in supraphysiological concentrations. 1,2 Recent animal studies with a mouse agonist suggest that CAR plays an important role in the metabolism of glucose, lipids, and fatty acids as well as in the endobiotic metabolism of bile acids, cholesterol, bilirubin, and thyroid hormones. 3 It has been proposed in several independent animal studies that CAR activation may ameliorate glucose homeostasis and insulin sensibility in the treatment of type 2 diabetes. 4,5 In addition, since CAR activation affects the expression of lipogenic genes in mice, this might also be a promising therapeutic intervention in the treatment of human obesity, steatosis, or hypercholesterolemia, 4,6−9 although contradictory and species-specific reports also exist. 10−12 CAR activators have been also proposed as a potential therapy for steatohepatitis or liver regeneration. 13,14 So far, only two human CAR crystal structures with a human agonist bound have been reported. 15 The CAR ligand-binding domain (LBD) cavity has a mostly hydrophobic and flexible character with a pocket size of 675 Å. 3,8,16 The hydrophobic cavity suggests that human CAR ligands are mostly highly lipophilic compounds.
Human CAR displays unique properties in comparison with other nuclear receptors as well as its rodent orthologues. CAR variant 1 (wtCAR, CAR1, and wild-type CAR) exhibits strong constitutive activity that can be further activated by agonists or repressed by inverse agonists. In addition, both direct LBDdependent and LBD-independent activation are known for CAR.
Human CAR is present in at least three transcript variants (wtCAR, CAR2, and CAR3) in the liver, which differ in their ligand-dependent activation and basal constitutive activities.  transcript variant X4), which has an insertion of the five amino acids APYLT into the LBD, represents 50% of transcripts. CAR3 has low constitutive activity but is highly inducible by ligands and much more active in the upregulation of CAR target genes in the liver. The transcript variant CAR2 (352 AA, NM_001077480.2) is a minor variant with moderate induction activity. The exact physiological functions of the variants are obscure, but several selective activators of individual variants have been described in the literature. 8,17−19 There are no highly potent, specific, and drug-like (with suitable physicochemical and ADME properties) agonists of the human CAR receptor without off-target effects that can be therapeutically used or can serve as a tool in therapeutic intervention with human CAR ligands. The unique properties of human CAR, mainly its hydrophobic pocket and high constitutive activity, make the discovery of specific ligands difficult. 20 Therefore, determining suitable drug candidate molecules targeting human CAR and high-affinity endogenous ligands remains problematic. 21 The only compound known to date is 6-(4-chlorophenyl)i m i d a z o [ 2 , 1 -b ] t h i a z o l e -5 -c a r b a l d e h y d e O -( 3 , 4dichlorobenzyl)oxime (CITCO, 1), which is a potent human�but not a mouse�CAR agonist. 22 However, this highly lipophilic compound also significantly activates the related pregnane X receptor (NR1I2, PXR) of the same subfamily through π−π interactions with the W299 residue. 22−24 This may exert an unfavorable effect on glycemia and liver steatosis. 25 On the contrary, the prototype mouse CAR ligand 1,4-bis[(3,5-dichloropyridine-2-yl)oxy]benzene (TCPOBOP) does not activate human CAR. 26 Different strategies have been used in high-content CAR ligand screenings recently performed, including nuclear translocation assays with an adenoviral-enhanced yellow fluorescent protein-tagged hCAR (Ad/EYFP-hCAR) vector in hepatocytes, 27,28 mammalian one-hybrid assays using a fusion protein of CAR or its LBD, 21,23,29−31 and assays employing stable luciferase reporter cell lines expressing wtCAR and treated with an inverse agonist, 32 as well as with a CAR3-selective screening method combined with other CAR assays. 33 In addition, studies employing pharmacophore computational modeling and the virtual screening of chemical databases have been performed. 30,34 In the past, several CAR activators with various structural features have been discovered in the screened libraries ( Figure  1A) or after modification of the lead compound CITCO (1), Figure 1B. 27−29,33−37 These human CAR ligands, however, still have limited potency to activate human CAR in nanomolar concentrations in comparison with the prototype high-affinity CAR ligand CITCO. Limited studies are currently being undertaken which explore structure−activity relationship variations by systematic synthesis on the human CAR ligand after the initial hit compound discovery or modification of the human CAR agonist CITCO as a template.

■ RESULTS AND DISCUSSION
We initially synthesized two analogues 2 and 3 of a known yet unspecific CAR agonist, CITCO, by modifying the middle flexible oxime linker to the triazole ring ( Figure 2). The oxime moiety is unstable under acidic conditions, which may complicate its use in vivo. The triazole ring offered stability, less flexibility, and good accessibility via an undemanding click reaction. Because of the synthetic feasibility and possibility to expand the variety of derivatives via a single reaction, the CuAAC reaction has been chosen. Different substitution patterns of blue, green, and red areas allowed us to explore the SAR of the compounds regarding the binding site, the bioavailability of the prepared compounds, and selectivity/ specificity toward the key receptors. Our decisions were also based on preliminary docking data (e.g., Figure S-3).
We found that analogues 2 and 3 significantly activate both CAR and PXR. Their activities and affinities toward human CAR were similar to those of CITCO in both the recombinant CAR LBD-dependent TR-FRET assay and cellular luciferase reporter assays. Their potency toward PXR was, however, more significant in comparison with the compound CITCO ( Figure 3). Compound 2 displayed less cytotoxicity in COS-1 cells than compound 3 (Table S-1).
Design and Synthesis of the First Generation of Novel CAR Ligands. The first modification of the phenyl and later benzyl ring led to two series A and B based on the compounds 2 and 3, respectively. The synthesis of the key compounds 2 and 3 as well as their modified analogues with a preserved triazole central heterocyclic linker is illustrated in Scheme 1.
Biology. The compounds with a substituted phenyl ring (12g−13i) appeared as potent agonists of the CAR with nanomolar EC 50 in a CAR TR-FRET assay. However, these compounds also significantly activated PXR, and most of them decreased the viability of COS-1 or HepG2 cells (Tables 2 and  S-1).
Compounds 14a−14f were also found as potent agonists of both CAR and PXR. Similarly, compounds 15a−h displayed significant activation of CAR and PXR with some moderate effects on cellular viability. Among these compounds, 15d was found as a highly efficient CAR agonist, while at the same time, it significantly activated PXR with E max higher than that of rifampicin ( Figure 4 and Table 2). Showing an opposite result, compounds 15i−m have marginal activities on the CAR and weak activity toward PXR (Table 2).
Compound 15i appeared to be a selective CAR ligand. However, its activity in the TR-FRET CAR coactivator assay was negligible, and its activity in the CAR LBD assembly assay was weaker compared to CITCO ( Figure 4). Thus, compound 15i demonstrates the phenomenon that some high-potency compounds in the TR-FRET assay with nanomolar EC 50 but less efficacy in cellular assays are, in fact, partial agonists of the CAR. These compounds do not reach the maximal activity (E max ) of full agonists such as CITCO or compound 15d ( Figure 4). We should also consider the possibility that the tested compounds are likely distributed into cell membranes in cellular assays, which results in lower potency (higher EC 50 in CAR AA and CAR3 assays) in comparison with the in vitro TR-FRET assay.
In contrast, compound 15j with a sulfonyl pyrrolidine moiety does not possess any activity to the CAR. We suppose that it is too bulky to fit into the CAR LBD domain (Table 2). We can conclude that the substitution of the phenyl ring with a lipophilic moiety increased activities for both the CAR and PXR. Similarly, lipophilic substitution or no substitution on the benzyl ring increased the nonselective activation of CAR and PXR. Compound 15d was found as an efficient dual CAR/ PXR agonist ( Figure 4). Interestingly, compounds 15f and 15h displayed high potency for wtCAR in TR-FRET and CAR AA assays (with EC 50 in the nanomolar range), but they were less  TR-FRET LanthaScreen CAR coactivation assay (CAR TR-FRET), CAR LBD assembly assay (CAR AA), luciferase reporter assay with CAR3 variant, or PXR-responsive luciferase reporter assay were used. b Compounds were tested at 1 μM (for the CAR3 assay) or at 10 μM for the PXR assay (n = 3). nd�not determined due to significant cytotoxicity (nd tox. ), due to extensive PXR activation or low CAR activation, nd # �not determined due to solubility problem and potential precipitation in solution. EC 50 is the concentration required to achieve half-maximum activation in the TR-FRET Lantha Screen CAR Coactivation assay or the CAR AA assay (in μM).
focused on the middle heterocyclic linker. The other rings (phenyl and benzyl) maintained the substitution pattern of compounds 2 and 3. The triazole ring was replaced by several heterocycles with one to three heteroatoms, such as thiadiazol or oxazole. The complete list is shown in Table 3.
Compounds 16A, 16B, 17, and 18 originated from the same precursors 25 or 26, which were synthesized by a condensation reaction of 4 or 5 with ethyl 3-(4-chlorophenyl)-3oxopropanoate in the presence of CBr 4 . The ethyl ester moiety of precursors 25 and 26 was hydrolyzed using LiOH· H 2 O, and the obtained acid derivatives 27 and 28 were treated with EDC and HOBt at 25°C, followed by the addition of substituted N′-hydroxyacetimidamide at 80°C to yield compounds 16A and 16B (Scheme 2). In order to synthesize compounds 17 and 18, the ester derivative 26 was reacted with an excess of hydrazine hydrate in EtOH, providing compound 29 and further acylated with 2-(3,4-dichlorophenyl)acetic acid by means of the peptide coupling reagent HATU in DMF. Ring-closing reaction of 30 with tosyl chloride at 25°C or Lawesson's reagent at 100°C overnight led to final compounds 17 and 18 respectively, although at very low yields (Scheme 2).
Thiazole analogues 19A and 19B were prepared from intermediates 6a and 7a, which were reacted with an excess of chloroacetyl chloride in dry dioxane at 70°C for 30 min and then heated up to 100°C overnight, followed by cyclization with ethanethioamide (1.5 equiv) in EtOH at reflux (Scheme 3).
Biology. When we tested the compounds with the modified middle heterocyclic linker, we found that the central moiety also contributes to both CAR and PXR activation, although there were no dramatic variations in the effects of different heterocycles. Out of the series of compounds, compound 19B showed higher relative selectivity to the CAR as it significantly activates CAR and CAR3, but it tends to activate PXR from 5 μM ( Figure 5). Similarly, compound 21 has high activity toward the CAR in the CAR LBD assembly assay, but it has a significant activity to PXR at a 1 μM concentration. Interestingly, in the CAR TR-FRET assay, the compound seems to be a partial agonist with the E max lower than that of CITCO or compound 19B (Table 4, Figure 5).
No significant cytotoxicity was observed for these compounds in COS-1 and HepG2 cells (Table S-2).
Design and Synthesis of the Third Generation of Novel CAR Ligands. Since the most promising biological results were found with compound 3 derivatives, in the next step, we addressed the modification of the benzyl ring with the main emphasis on the meta position while maintaining the triazole ring. The chlorine atom was replaced with a series of acyl molecules (Table 5). This resulted in improved estimated water solubility and bioavailability of the compounds.
The key compound 37 was prepared by the same means as compound 3 (Scheme 1) with a minor modification in the azide coupling partner. Straightforward hydrolysis led to acid analogue 38, which was converted to amide analogues 39−42 via acylchloride 52 (Scheme 6).
In order to increase bioavailability, compound 39 was converted to its HCl salt 39 HCl (Scheme 6). The subsequent reaction of N-methoxy-N-methylbenzamide derivative 42 with a Grignard reagent or LAH at low temperature yielded ketone (43,44) or aldehyde analogues 45, which upon the reaction with hydroxylamine provided derivative 46. Finally, the reduction of the ester derivative with subsequent methylation provided compounds 47 and 48 (Scheme 6).
N-acyl derivative 51 was synthesized in two steps from nitro derivative 49 after reduction and the succeeding acylation reaction (Scheme 7).
Next, we decided to broaden the number of examples of heterocyclic linkers with six-membered heterocycle pyridine  a TR-FRET LanthaScreen CAR Coactivation assay (CAR TR-FRET), CAR LBD assembly assay (CAR AA), luciferase reporter assay with CAR3 variant, or PXR-responsive luciferase gene assay were used. b Compounds were tested at 1 μM (for CAR3 assay) or at 10 μM for PXR assays (n = 3). nd�not determined due to significant cytotoxicity (nd tox. ), due to extensive PXR activation, or low CAR activation (nd). EC 50 is the concentration required to achieve halfmaximum activation in the TR-FRET LanthaScreen CAR coactivation assay or CAR AA assay (in μM). and to replace the original triazole with an aryl ring. Syntheses of these compounds started from iodinated precursor 9a, which was coupled with aryl/pyridyl boronic acid (Scheme 8), followed by the Negishi coupling reaction with benzylzinc bromide catalyzed by Pd, providing compounds 55 and 56, respectively. Unfortunately, these compounds were barely soluble, so we did not test them. Moreover, the methylene part of the linker was exchanged for O and NH. Similarly, to the previously mentioned compounds, intermediate 9a was coupled with appropriate boronic acid (Scheme 9), providing intermediate compounds 57 and 58 with a free amino group and methoxy group, respectively. Demethylation of compound 58 afforded compound 59 with a free hydroxy group. Both compounds 57 and 59 were coupled with 5-bromo-2-chlorobenzamide by way of Buchwald or Ullmann coupling conditions, yielding compounds 60 and 61 (Scheme 9, Table 6).
Biology. When we tested derivatives of compound 3 (Series B) with acyl moieties in the meta position of the benzyl ring with the preserved triazole ring as a linker (37−44), we found that the moieties significantly contribute to CAR activation but not to PXR activation. Carboxylic acid itself in the meta position (38) resulted in a complete loss of CAR activation. However, amides, as well as esters, significantly activated CAR in all assays. Compound 37 appeared as the most efficient to activate the CAR in the TR-FRET LanthaScreen CAR coactivation assay and highly efficient to activate a CAR LBD assembly assay with an EC 50 lower than that of CITCO (EC 50 = 0.4 and 152 nM vs 12 and 690 nM, respectively). Importantly, a methylester (compound 37), amides (39, 40, and 41) as well as N-methoxy-N-methylamide (compound 42) all have minimal (37 and 41) or no activity (39, 40, and 42) to activate PXR at 10 μM ( Figure 6 and Table 7). Other compounds from the set also display low activation of PXR. These compounds were also noncytotoxic in viability assays (Table S-3).
When we looked in detail at the CAR agonists 39, 40, 41, and 42 without significant PXR activation, their potencies in the TR-FRET LanthaScreen CAR coactivation assay were by an order of magnitude lower (and EC 50 higher) than that of CITCO and compound 39 seems to be a partial agonist of the CAR in the assay. In the case of CAR LBD assembly and CAR3 variant assays, these compounds activated the CAR LBD with lower but still comparable affinities in comparison with CITCO. This phenomenon may be explained by the different activation of the CAR LBD by these compounds via another coactivator than with PGC1α, which is involved in the TR-FRET CAR coactivation assay. Indeed, SRC-1 (NCOA1)  along with other coactivators are important for the coactivation of CAR 8 and we can suppose an array of different coactivators in CAR variants activation in cellular assays. We may also suppose the intracellular accumulation of these compounds, for example, via an uptake mechanism, which may increase their potencies in cellular CAR LBD assembly and CAR3 variant assays, but not in the TR-FRET LanthaScreen CAR coactivation assay.
Compounds with substitution of the meta position of the benzyl ring with other substituents (43−51) and with the preserved triazole ring as a linker retained efficient CAR activation with high potency (EC 50 below 0.1 μM) ( Table 7), although these compounds also activate PXR to some degree. Some of these display substantial effects on cellular viability (Table S-3), which may affect cellular assays. With a methoxyethyl moiety, compound 48 was found to activate the CAR LBD assembly assay with the lowest EC 50 = 24.9 nM; however, the compound is not selective for the CAR and significantly activates PXR (EC 50 = 4.34 ± 1 μM). Compound 48 was also highly potent in the activation of the CAR3 variant in the CAR3 variant assay (Table 7). Interestingly, some compounds such as 45 and 47 had high potency for wtCAR in the TR-FRET and CAR AA assays, but they were less potent in the CAR3 assay, suggesting some selectivity for the wtCAR variant.
Compounds 60−61 with a replaced methylene part of the linker with O and NH (Table 6) lost the activity to the CAR and retained a weak activity to PXR.  Yields in % correspond to the last reaction step after purification.

Interactions with Human CAR Variants.
Next, we decided to analyze our novel selective CAR agonists 37, 39, 40, 41, and 42 to determine whether they could upregulate CYP2B6 gene mRNA, the typical CAR target gene, in PHH from one donor. We found that all compounds could significantly upregulate CYP2B6 mRNA. Compounds 39 and 42 tend to be the most potent with a 1 μM concentration in the experiments. These data suggest that the compounds are metabolically stable in metabolically competent hepatocyte cells and that they enter hepatocytes to activate the CAR ( Figure 7A).
In translocation experiments with the EGFP-hCAR + Ala chimera, we examined the tested compounds to determine whether they stimulate cytoplasm-to-nuclear translocation of the activated human CAR with extra alanine in the LBD (CAR + A). 42 We noted that mainly CITCO and compounds 37, 39, and 40 significantly decrease the number of cells with specific cytoplasm EGFP-hCAR + Ala localization, and they increase the portion of cells with nuclear localization of the CAR chimera (Figures 7B and S-1). In these experiments, compound 39 appeared as the most promising candidate.
In agreement with cellular assays or induction experiments in PHHs, tested compounds have similar activities in comparison with CITCO (comp. 1) or compound 37, which are high-affinity CAR agonists in TR-FRET CAR assays. These data suggest that the cellular environment and signaling have a significant determination on CAR activation.
In the next experiments, we sought to determine whether the discovered selective CAR agonists 37, 39, 40, 41, and 42 interact with wild-type CAR (wtCAR), human CAR variants 2 (CAR2) and 3 (CAR3), as well as with mouse CAR orthologue in luciferase reporter assays. Efficacy to activate wtCAR was assessed using the CAR LBD assembly assay (CAR AA) or with a wtCAR expression vector that was inhibited with PK111195 (0.1 μM), a known CAR inhibitor. We found that compound 37 is highly efficient in the stimulation of the variant CAR2 and other variants of CAR in comparison with CITCO (100% activity). Compound 39 significantly activated wtCAR in the CAR LBD assembly assay and the CAR3 variant in the gene reporter assay. Its activity in the assay with wtCAR and its inhibitor PK11195, however, was low, suggesting a weak efficacy to compete with the PK111195 inhibitor in the CAR LBD. Other candidate compounds have lower potency in comparison to CITCO (100% activity) in the activation of CAR variants. Compound 42 appeared as a combined agonist of wtCAR and its variants in all assays. Only compound 37 was found to stimulate the mouse CAR when compared to the mouse ligand TCPOBOP ( Figure 7C).
In the follow-up studies, we examined the stability of compound 37 in human and mouse microsomes and in plasma. We found that compound 37 is unstable in both mouse and human microsomes with t 1/2 = 4.78 ± 1.31 min and t 1/2 = 6.38 ± 0.67 min, respectively. Importantly, we found that compound 37 is also unstable in mouse plasma as well with t 1/2 = 22.76 ± 0.03 min ( Figure S-2).

Selection of the Candidate for Animal Studies and Detailed Characterization of Compound 39.
In the next experiments, we studied the most efficient compound 39 in five PHHs from five different donors to determine whether it could upregulate CYP2B6, CYP3A4, and CYP2C9 mRNA. These genes are significantly, but not exclusively, regulated via the CAR in human hepatocytes. Despite high variability in response in different hepatocyte preparations, we found that compound 39 has similar activity to induce these genes in comparison with CITCO ( Figure 7D). Western blotting experiments in PHHs (BioIVT) treated with comp. 39, rifampicin, CITCO, and PXR antagonist SPA70 (10 μM) for Compounds were tested at 1 μM (for CAR3 assay) or at 10 μM for PXR assays (n = 3). nd�not determined due to significant cytotoxicity (nd tox. ), due to extensive PXR activation, or low CAR activation (nd). EC 50 is the concentration required to achieve half-maximum activation in the TR-FRET LanthaScreen CAR coactivation assay or CAR LBD assay (in μM).
48 h revealed that compound 39 up-regulates CYP2B6 protein and that the upregulation is not abolished by the PXR antagonist SPA70 ( Figure 7D, inserted panel).
To confirm that compound 39 induces CYP2B6 mRNA via the activated CAR, we performed experiments with HepaRG and its KO CAR counterpart cell line without CAR expression. We observed the upregulation of CYP2B6 and CYP3A4 mRNA only in the HepaRG cells but not in the HepaRG KO CAR cells after treatment with both CITCO and compound 39 ( Figure 7E,F).
In the next experiments, CYP2B6 and CYP3A4 mRNA expression were analyzed in LS174T cells using RT-qPCR. LS174T cells express endogenous PXR but lack functional CAR. 43 We did not observe any significant induction of these genes by compound 39 in these cells ( Figure 7G).
Then, we performed luciferase gene reporter assays with the CYP3A4 gene promoter construct (p3A4-luc) in HepG2 cells transfected with either PXR or CAR3 expression constructs. Compound 39 activated the luciferase construct only in the presence of CAR3, and the PXR antagonist SPA70 had no significant effect on the activation ( Figure 7H). Finally, we examined the dose−response activation of CAR2 and CAR3 variants with compound 39 ( Figure 7I). Unfortunately, the profiles of the dose−response curves did not reach the plateau phase and did not allow us to calculate EC 50 and E max values in the range of concentrations up to 30 μM ( Figure 7I).
Based on the data, we can conclude that compound 37 is the most active ligand for all CAR variants. Compound 39 displayed the most significant activity in the induction experiments in PHH and HepaRG cells irrespective of their lower affinities to wtCAR or CAR3 variants in CAR TR-FRET and cellular assays as well as marginal activity toward the CAR2 variant. In addition, we found that compound 39 does not induce CYP2B6 or CYP3A4 mRNA via PXR activation.
Next, we considered the physicochemical properties of selected compounds such as molecular weight (M w ), Log S (the solubility of a substance, measured in mol/L), and Log P (the partition coefficient is a ratio of concentrations of nonionized compound between water and octanol). Compound 39 is the smallest and less lipophilic candidate compound with better-predicted water solubility among the selected candidate compounds (Table 8).
Therefore, we decided to use compound 39 in further experiments with other nuclear receptor assays and in humanized CAR mice.
Novel CAR Ligands Interact with His203 and Occupy a Hydrophobic Pocket in Human wtCAR-LBD. For the modeling analyses, we docked the compounds 37, 39, 40, and 48 in human wtCAR-LBD using CITCO as a reference ( Figure  8A,B). Furthermore, to explore the wtCAR-LBD conformational dynamics and the interactions of these novel compounds, we conducted 25 μs of all-atom MD simulations (5 μs for each system plus CITCO). We studied the differences in the protein−ligand interactions among the systems, in comparison to CITCO. We observed the relevant role of the hydrogen bond interaction of H203 with the phenylimidazole ring in the novel compounds ranging from ∼65 to 90%. This interaction was observed in particular for compounds 39 and 40 for ∼65 and 90%, respectively . It is noteworthy that in the wtCAR/ CITCO simulations, these additional polar interactions are not observed.
In addition to the hydrogen bond interactions, all novel compounds show high hydrophobic interaction frequency with F161 (∼100%), the H203 imidazole ring (∼70 to 100%), and Y224 (90−100%, except for the compound 40, which is around 20%), and lower interaction with C202, F234, Y326, and L242 (no interaction with compound 40) ( Figure 8C,D; Figures S-7 and S-8). These interactions were similarly observed with CITCO. Also, some interactions are compound-specific such as I164 with compound 39, L206 with compounds 37 and 40, F217 with compounds 37, 40, and 48, and L239 with compounds 40 and 48. Overall, we observed that all the novel compounds adopted U-shaped conformations similar to CITCO within the wtCAR-LBD ( Figure S-6). This conformation is mainly supported by hydrophobic interactions, with an exclusive interaction for compound 39 with I164, and extra T225 and D228 hydrogen bonds for compound 39, which stabilizes the compound within human wtCAR-LBD.
H12 Positioned in Close Vicinity of H3. MDs revealed no direct interaction between CITCO and residues from H12. In this regard, we then proceeded to investigate the changes in geometry and dynamicity of this region relative to the LBD with novel compounds and CITCO. For this purpose, we calculated the distance between H12 and H3 (center of mass of each helix). The result showed that all novel compounds can stabilize the conformation of H12 in the close vicinity of H3 similar to CITCO ( Figure 8E, Figure S-9A). This geometry is known to initiate receptor activation. 15 It has been reported that H12 stays away from the pocket due to the barrier formed by hydrophobic residues in the LBD, 44 where H11 directs the H12 in this active position. 15 Previous studies also indicate that the free carboxylate of the H12 C-terminus interacts with the K195 side chain (on H4), leading to further H12 stabilization. 15 To assess this phenomenon over the simulation time, we next calculated the distance between the carboxylate group of the H12 C-terminus and the polar group of K195 ( Figure 8E; Figure S-9B). The median value for this distance in both wtCAR/CITCO and wtCAR/compounds 37, 39, and 40 stands around 3.1 Å, with a further distribution with compound 48. This geometry enables the hydrogen bond formation between the H3 and H12 regions, providing extra stability to the systems. Taken together, this supports our result in terms of the high binding affinity and potency of our novel compounds.
Further Geometry Stabilization through N165−Y326 Interaction. Along with the closeness of H12 and H3, and the interaction between the H4 and H12 C-terminus, the stabilization of the systems comes through the hydrogen bond interaction between N165 (H3) and Y326 (H10). Both CITCO and 39 show relatively similar rigidity in this region ( Figure 8F). The same trends are also observed with other novel compounds (Figure S-8C) with further distribution in the presence of compounds 40 and 48. Although this interaction has been previously observed in the crystal structure with CITCO, 15 MD data indicates that it is also relevant for our novel compounds.
Taken together, our docking data followed by microsecond timescale all-atom MD simulations revealed that CITCO and compound 39 interact with wtCAR-LBD mainly by hydrophobic contacts and that stronger polar contacts were formed between compound 39 and wtCAR-LBD compared to CITCO due to hydrogen bond interactions between comp. 39 amide moiety and T225 and D228 backbone oxygen. Interestingly, previous findings report that no specific hydrogen bonds are required for CITCO stability inside the CAR. 18,45 Analyses of the MD trajectories showed that the interaction between compound 39 and I164 besides the higher interaction frequency with Y326 (hydrophobic interaction) compared to that of the CITCO ( Figure 8D) could highlight the critical role of H3 and H10 in protein stabilization. Of note, H10 lies on the heterodimerization interface where RXRα binds to the CAR. Our MD data also revealed that the H12 region is ordered and stable upon ligand binding. This event has been earlier reported as a driving force for CAR constitutive activity 15 and, therefore, supports the agonistic effect of compound 39.

Selectivity of Compound 39 to Other Nuclear
Receptors. Next, we sought to determine whether compound 39 is selective to the human CAR and whether it activates other nuclear receptors, for which a set of luciferase reporter assays was employed. We confirmed the selectivity of compound 39 for CAR as with no other nuclear receptor or the transcription factor aryl hydrocarbon receptor (AhR) was significantly activated by the compound at 10 μM concentration ( Figure 9).
Microsomal Stability Experiments and Pilot Animal Pharmacokinetic Study. In the following experiments, we evaluated both the plasma and microsomal stability of compound 39 HCl in human plasma, human liver microsomes, as well as liver fraction S9 in time intervals of up to 120 min ( Figure 10A,B; Table S-5). We found that compound 39 is highly stable in human plasma (t 1/2 ≥ 240 min). However, we observed a significant decline of compound 39 concentration in human microsomes as well as fraction S9 (t 1/2 = 38.04 min and t 1/2 = 42.4 min, respectively) ( Figure 10B; Table S-5).
We also evaluated the plasma protein binding of compound 39 in both human and mouse plasma, determining that 98% of compound 39 is bound to human plasma proteins (Table S-6). We observed very similar properties of compound 39 in mouse plasma and mouse hepatic microsomes (Tables S-5−S-7).
In a pilot single-dose pharmacokinetic study, we found fast absorption of compound 39 HCl hydrochloric salt after p.o. application in gavage, although the compound was rapidly eliminated from the plasma ( Figure 10C; Table S-9). Significantly, we detected traces of metabolites for compound 39 after i.v. application in plasma. Metabolites M1 and M2 represent compounds 41 and 40. Both compounds are Nmethylated derivatives of compound 39 with significant CAR activity. Minor metabolite M3 (compound 34) is 2-(4chlorophenyl)-3-(1H-1,2,3-triazol-4-yl)imidazo[1,2-a]pyridine, indicating that hepatic metabolic enzymes may attack the methylene bridge between the heterocycle and phenyl rings ( Figure 10D). The metabolite is inactive with respect to the CAR activation, and the metabolite was not observed in human liver microsomes with the S9 fraction (data not shown). These data suggest that compound 39 is the main active compound, and it is likely eliminated intact as the parent compound. Nevertheless, further detailed pharmacokinetic studies should focus on the distribution, biliary elimination, and phase II metabolic clearance of the compound.
In addition, we conducted another examination to determine whether compound 39 inhibits the activities of major human cytochrome P450 enzymes. We found that compound 39 has a minor effect on major cytochrome P450 enzymes. Compound 39 inhibits enzymatic activities of CYP3A4 (with IC 50 = 16.08 μM) and CYP1A2 (IC 50 = 21.07 μM) in higher micromolar concentrations, but the compound has no activity on the CYP2B6 enzyme up to 30 μM concentration ( Figure 10E).
Effects of Compound 39 in CAR Humanized Mice. Next, we treated humanized PXR/CAR/CYP3A mice with compound 39 to study the regulation of CAR target genes after a single i.p. application.
We found that compound 39 significantly upregulates Cyp2b10 mRNA and protein, and human CYP3A4 mRNA in the humanized model, but significantly decreases the expression of genes Scd1 and G6pc after a single dose of 1 mg/kg. The latter genes are critically involved in triglyceride synthesis and gluconeogenesis in the liver. CITCO appeared more potent to induce Cyp2b10 mRNA but less potent to upregulate CYP3A4 mRNA expression, confirming the high efficiency of compound 39 to regulate the key CAR targets genes in murine hepatocytes. We also observed the trend of a decrease of Srebp1 and Fasn mRNA expression after compound 39 application (1 mg/kg) ( Figure 11A), which agrees with data observed with the mouse CAR ligand TCPOBOP. This suggests that the human CAR ligand 39 recapitulates the significant effect of the murine ligand TCPOBOP on the regulation of lipid metabolism. 4,5 We did not observe upregulation of the genes involved in rodent liver proliferation after CAR activation and liver weight gain in the experiments ( Figure 11B,C). Nevertheless, longterm studies are needed to examine liver hypertrophy and hyperplasia after repeated treatment with compound 39.
In analyzing blood biochemistry data after the single-dose application of compound 39 (dose 10 mg/kg), we observed a statistically significant decrease in plasma low-density lipoprotein (LDL) levels. This is consistent with results found with the mouse CAR ligand TCPOBOP in wild-type mice, indicating a positive effect of CAR activation on LDL plasma levels. 9 We also observed a decrease in bile acid and total bilirubin (bilirubin-T) plasma levels after the application of compound 39, although these effects were not statistically significant. Neither glucose, plasma triglycerides (TG), HDL lipoproteins, nor liver injury biomarkers (AST, ALT, and LDH) was significantly affected by compound 39 after the single-dose application ( Figure 11D).
These results of the pilot single-dose pharmacokinetic study suggest that compound 39 is a novel effective human CAR agonist in animal experiments, a finding which warrants further repeated-dose long-term proof-of-concept studies.
Toxicity Studies of Compound 39. We observed no cytotoxicity in HepG2, COS-1 (Table S-3), HepaRG, HepaRG KO CAR, or in the PHHs after 48 h treatment (data not presented). Furthermore, in the Repeated Dose 7 day Oral Toxicity Study in Rodents (EMA/CPMP/ICH/286/1995, 2009 guidelines), no significant signs of toxicity were observed after the 7 days of oral administration of compound 39 HCl into rats. In particular, no significant changes in body weight, changes in behavior, gross pathology, hematology, and biochemistry parameters were observed after the 7 days of oral administration of the compound 39 HCl in all groups (groups with 1, 10, and 30 mg/kg b.w.) when compared to the control group. We also tested the cardiotoxicity of compound 39 in a modified hERG fluorescence polarization assay. We did not observe any binding of compound 39 to hERG up to 20 μM (Supporting Information, Chapter 10).
Finally, we did not observe any frame-shift or base-pair substitution mutagenicity of compound 39 in a modified Ames fluctuation assay performed on Salmonella typhimurium TA100 and TA98 strains at a concentration of 1 and 10 μM (Table S-10).

■ CONCLUSIONS
Attempts to delineate the therapeutic implications of the CAR in humans have been hindered by the significant overlap in the pharmacology of human CAR and PXR receptors and the lack of a highly selective and potent human CAR agonist with suitable ADME properties.
In this work, we used a rational design of novel selective human CAR agonists. We applied a bioisosteric approach to the central part of the hit molecules 2 and 3 to prepare new ligands for this human nuclear receptor. We were thus able to design a series of novel compounds that differed significantly in both nominal activities as CAR agonists and selectivity toward the PXR receptor as well as enhanced stability in comparison with the model compound CITCO. Based on our results, we performed a careful multiparametric selection of suitable candidates for further pharmacodynamic and pharmacokinetic studies. We found that the imidazo[1,2-a]pyridine core with the 1,2,3-triazole linker can be used for the further design of specific human CAR ligands. Replacement of the flexible oxime linker of CITCO with the triazole ring offered stability, less flexibility, and good accessibility via an undemanding click reaction. Modification of the 3,4-dichlorphenyl moiety of the hit compound 3 with amides (analogues 39−42) resulted in CAR ligands without agonistic activities to PXR (Scheme 6, Table 7, and Figure 6). Although extremely potent CAR agonists emerged in the resulting library of compounds, we also had to consider their metabolic stability and activity toward PXR. As a result, we decided to use compound 39 for further experiments, which, although not among our most potent CAR agonists, exhibited a desirable CAR/PXR profile and reasonable metabolic stability, allowing subsequent in vivo experiments. Using this chemical tool, which we have shown to have no observable toxicity or genotoxic potential, we were able to prove that compound 39 significantly activates the human CAR, both in vitro in human hepatocyte models and CAR humanized mice. Significantly, we noted that compound 39 regulates typical CAR target genes involved in xenobiotic (Cyp2b10), lipid (Scd1), or glucose (G6pc) metabolism, and it Figure 11. In vivo effects of compound 39 on liver CAR target genes involved in the intermediary metabolism of glucose, lipids and bile acids, hepatocyte proliferation, and apoptosis in humanized PXR/CAR/CYP3A mice (n = 4) after single i.p. application of the dose 1 or 10 mg/kg. Mice were sacrificed 36 h after application; livers were subjected to RT-qPCR analysis (A,B), western blotting analysis with anti-Cyp2b10 antibody, or were weighted (C). Blood samples were analyzed for biochemical parameters (D). *p < 0.05-significant effect vs control (vehicle-treated) mice. decreases plasma LDL lipoproteins even after a single dose in humanized PXR/CAR/CYP3A4/3A7 mice.
In summary, our work identifies a selective CAR receptor agonist for which we have demonstrated both in vitro and in vivo activities in relevant models for the human CAR. Compound 39 thus warrants further preclinical studies in humanized CAR models or human hepatocyte models to better understand the unique function of the human CAR without confounding off-target effects on PXR receptor activation.
■ METHODS Experimental Methods. Synthesis of Novel Ligands. General chemical procedures: NMR spectra were measured on a Bruker AVANCE II-600 and/or Bruker AVANCE II-500 instruments (600.1 or 500.0 MHz for 1 H and 150.9 or 125.7 MHz for 13 C) in hexadeuterodimethyl sulfoxide and referenced to the solvent signal (δ 2.50 and 39.70, respectively). Mass spectra were measured on a LTQ Orbitrap XL (Thermo Fischer Scientific) using electrospray ionization (ESI) and a GCT Premier (Waters) using EI. The elemental analyses were obtained on a Perkin Elmer CHN Analyzer 2400, Series II Sys (PerkinElmer), and X-ray fluorescence spectrometer SPECTRO iQ II (SPECTRO Analytical Instruments, Germany). Column chromatography and thin-layer chromatography (TLC) were performed using Silica gel 60 (Fluka) and Silufol Silica gel 60 F 254 foils (Merck), respectively. The purity of newly synthesized compounds was >95%, confirmed by UPLC-MS. Solvents were evaporated at 2 kPa and a bath temperature of 30−60°C. The compounds were dried at 13 Pa and 50°C.
General Procedure I: Cyclization of Heterocycle. 2-Aminothiazole (3) or 2-aminopyridine (4) was dissolved in EtOH, and substituted or unsubstituted bromoacetophenone derivative (1 equiv) was added, followed by the addition of NaHCO 3 (1 equiv). The reaction mixture was heated at 70°C overnight. After the completion of the reaction (monitored by TLC or UPLC), the solvent was evaporated to a minimal volume, and the residue was diluted with EtOAc and washed with water. The water phase was extracted twice more with EtOAc, and the combined organic phases were dried over sodium sulfate and evaporated. The residue was purified by flash column chromatography (eluent petrol ether/EtOAc or EtOAc/MeOH).
General Procedure II: Iodination. 2-Substituted imidazo[1,2a]pyridines or imidazo[2,1-b]thiazoles were dissolved in CH 3 CN (5 mL/mmol) and NIS (1.05 equiv) was added in one portion. The suspension was stirred at 25°C, and the conversion was monitored by TLC. After the completion of the reaction (1−4 h), the reaction mixture was diluted with EtOAc and washed with a saturated Na 2 S 2 O 3 solution. The inorganic phase was extracted twice more with EtOAc; combined organic phases were dried over sodium sulfate and evaporated. The residue was purified by flash column chromatography, with the mobile phase petrol ether/EtOAc (10:50%).
General Procedure III: Sonogashira Coupling. 3-Iodoimidazo[1,2a]pyridines or 5-iodoimidazo[2,1-b]thiazoles were placed in a dried round-bottom flask, diluted with dry DMF, degassed at 0°C, and flushed with argon. CuI (10 mol %) and Pd(PPh 3 ) 2 Cl 2 (5 mol %) were added, the mixture was properly degassed, dry TEA (3 equiv) was added, and the mixture was degassed again. Finally, TMSacetylene (5 equiv) was added in one portion. The reaction mixture was stirred at 25°C under an argon atmosphere. After the completion of the reaction (monitored by TLC), the mixture was diluted if necessary with CHCl 3 and filtered over Celite. The filtrate was washed with water; the water phase was extracted twice more with CHCl 3 ; the combined organic phases were dried over sodium sulfate and evaporated. The residue was purified by flash column chromatography (mobile phase petrol ether/EtOAc).
General Procedure IV: Click Reaction. Trimethylsilyl(ethynyl)imidazo[2,1-b]thiazole or trimethylsilyl(ethynyl)imidazo[1,2-a]pyridine derivatives were dissolved in THF/H 2 O mixture (1:1) and an appropriate azido intermediate (1 equiv) was added. The reaction mixture was degassed at 0°C, refilled with argon, and CuSO 4 ·5H 2 O (10 mol %), KF (1 equiv), were Na-ascorbate (1 equiv) were added in one portion. The reaction mixture was stirred at 25°C and monitored by TLC. After the completion of the reaction, the mixture was diluted with EtOAc and washed with water. The water phase was extracted twice more with EtOAc; combined organic phases were dried over sodium sulfate and evaporated. The residue was purified by flash column chromatography (eluent petrol ether/EtOAc or EtOAc/ MeOH).
General Procedure V: Ester Hydrolysis. The methyl or ethyl ester derivative was dissolved in THF/H 2 O 2:1, and LiOH·H 2 O (4 equiv) was added in one portion. The reaction mixture was stirred at 25°C and monitored by TLC. After the completion of the reaction, the mixture was extracted with EtOAc, and the water phase was acidified to pH 2 and extracted again with EtOAc. The organic phase was dried over sodium sulfate and purified by reverse-phase flash column chromatography.
In Silico Molecular Dynamics Analysis. Molecular Modeling. Receptor and Ligand Preparation: The crystal structure of the hCAR model was retrieved from the RCSB Protein Data Bank (www.rcsb. org) (PDB code: 1XVP). 15 All ligands for docking were drawn using Maestro (2020.2) and prepared using LigPrep to generate the three-dimensional conformation, adjust the protonation state to physiological pH (7.4), and calculate the partial atomic charges, with the force-field OPLS3e. We employed a standard docking to accommodate the compounds 37, 39, 40, and 48 within the CAR's LBD (PDB ID: 1XVP; resolution: 2.0 Å, cocrystallized with compound 1, 15 amino acid numbering follows the crystal structure), using Glide. 57 Ligands were docked within a grid around 12 Å from the centroid of the cocrystallized ligand generating 10 poses per ligand. To validate the docking obtained for test ligands, and also to evaluate the capability of the docking algorithm to locate the ligands within the LBD, we redocked the cocrystal ligand (compound 1, a full agonist) inside the CAR LBD. Next, the seven systems (four test compounds plus compound 1) were prepared and minimized by adding hydrogens, adjusting the protonation states of amino acids, and fixing missing side-chain atoms and protein loops using Maestro PrepWizard 2020.2. The molecular dynamics simulation protocol and respective analyses can be found in the Supporting Information, Chapter 2. For each ligand, simulations of five 1 μs independent replicas were carried out, resulting in 25 μs worth of simulations for all five systems.
Statistical Analysis. Data are presented as the means and SD from at least three independent experiments (n = 3). A one-way analysis of variance (ANOVA) with Dunnett's post hoc test was applied. GraphPad Prism ver. 9.3.1. Software (GraphPad Software, Inc., San Diego, CA, United States) was used to perform statistical analysis.
EC 50 indicates the xenobiotic concentration required to achieve half-maximum activation, and relative E max represents the overall maximal calculated activation produced by the tested compound (i.e., maximal efficacy). The activities of compound 1 and rifampicin at 10 μM were set to be 100% in the dose−response calculations. IC 50 represents the half-maximal inhibitory concentration in the viability MTT assay or in cytochrome P450 inhibition assays. A p-value of <0.05 was considered to be statistically significant.